1. Field of the Invention
This invention relates to compositions and assay methods for the extraction and hybridization of nucleic acids. In particular, this invention relates to compositions and methods to extract nucleic acids from cells in complex biological samples or specimens without the use of toxic compounds, such as phenol and/or chloroform, primarily through the use of benzyl alcohol or benzyl alcohol derivatives. The novel compositions and methods described here are very effective in the extraction and purification of nucleic acids.
2. Brief Description of the Relevant Art
Organic solvents such as phenol and chloroform are traditionally used in techniques employed to isolate nucleic acid from procaryotic and eucaryotic cells or from complex biological samples. Nucleic acid isolations typically begin with an enzymatic digest performed with proteases followed by cell lysis using ionic detergents, and then extraction with phenol or a phenol/chloroform combination. The organic and aqueous phases are separated and nucleic acid which has partitioned into the aqueous phase is recovered by precipitation with alcohol. However, phenol or a phenol/chloroform mixture is corrosive to human skin and is considered as hazardous waste, which must be carefully handled and properly discarded. Further, the extraction method is time consuming and laborious. Marmur, J. Mol. Biol. 3:208-218 (1961), describes the standard preparative procedure for extraction and purification of intact high molecular weight DNA from procaryotic organisms using enzymatic treatment, addition of a detergent, and the use of an organic solvent such as phenol or phenol/chloroform. Chirgwin et al., Biochemistry 18:5294-5299 (1979) described the isolation of intact RNA from tissues enriched in ribonuclease by homogenization in guanidinium thiocyanate and 2-mercaptoethanol, followed by ethanol precipitation or by sedimentation through cesium chloride.
Further, the use of chaotropic agents such as guanidinium thiocyanate (GnSCN) are widely used to lyse and release nucleic acid from cells into solution, largely due to the fact that chaotropic salts inhibit nucleases and proteases. However, it has proved difficult to isolate the nucleic acids from these chaotropic salt solutions due to the incompatibility of the chaotropes with ionic detergents and the inability to easily partition the nucleic acid into an aqueous phase, given the use of such high molar concentrations of salt.
The ability to effectively inhibit nucleases during nucleic acid isolation procedures is paramount, especially when the starting material is complex, such as feces or blood. In 1959, Brownhill et al. reported that bentonite was an inhibitor of nucleases (Brownhill et al., Biochem. J. 73:434 (1959)). Fraenkel-Conrat et al. later developed a procedure for the use of bentonite to inhibit ribonuclease in a procedure to purify tobacco mosaic virus (Fraenkel-Conrat et al., Virology 14:54-58 (1961)). Subsequent researchers reported the use of bentonite in combination with phenol and chloroform in the reduction of ribonuclease activity during the isolation of RNA (Jacoli et al., Can. J. Biochem. 51:1558-1565 (1973); Griffin et al., Anal. Biochem. 87:506-520 (1978); Grady et al., Anal. Biochem. 101:118-122 (1980)). It has also been reported that DNase 1 and .alpha.-amylase can be made RNAse-free by treatment with bentonite (Garrett et al., Anal. Biochem. 52:342-348 (1973)).
All of the above traditional nucleic acid extraction procedures, thus, require the use of toxic compounds such as phenol and/or chloroform. Researchers have long sought safer effective extraction procedures.